Project Summary Adult tissues possess long-lived stem cells (SCs) that are responsible for maintaining and repairing their lineage. Melanocyte stem cells (McSCs) are unique adult SCs located in hair follicles that maintain the population of differentiated, pigment-producing melanocytes. To function properly, the balance between SC quiescence, activation, and differentiation must be tightly regulated, and McSCs cycle between these states in sync with the hair cycle. Understanding how McSCs transition from quiescence to activation and eventual differentiation may provide insight that could be used to develop new treatments for melanocyte-related pathologies. These include conditions such as the autoimmune pigmentation disorder vitiligo, where stimulating quiescent McSCs with ultraviolet radiation has been shown to restore pigment-producing progeny to the skin. Additionally, with increasing evidence suggesting that cancers hijack fundamental SC mechanisms, this information will be important for understanding and treating melanoma. Therefore, the objective of my proposed study is to elucidate the gene expression programs that drive McSC quiescence and differentiation using comparative transcriptional profiling and functional testing in vivo. My preliminary data indicate that there is significant differential gene expression between quiescent McSCs and their differentiated progeny, many of which are transcription factors (TFs) with no known role in McSC biology. My central hypothesis is that distinct transcriptional programs fueled by specific TFs are responsible for maintaining quiescence and activation/differentiation of McSCs. In this study, I will 1) mechanistically define the TF genes that promote McSC quiescence, and 2) interrogate key genes of the mature melanocyte signature for their role in driving activation/differentiation. Using bioinformatics and pathways analysis, I have selected TF candidates that I hypothesize are key regulators of each process. To start, I will map the temporal expression patterns of each factor in their native microenvironment using in situ hybridization and immunofluorescence staining of back skin throughout hair cycling. TFs will then be manipulated in vitro to identify the most promising candidates for in vivo testing. For this purpose, I have developed an approach combining CRISPR/Cas9 technology with our lab's well-established in utero lentiviral delivery system. Lentivirus containing sgRNA will be used to transduce the neural crest (from which McSCs and melanocytes arise) of embryos with lineage-specific and inducible TyrCreERT2-dependent Cas9 expression. Tamoxifen will be administered postnatally to activate Cre, turn on Cas9, and knockout my candidate. After ablating these TFs, I will assess the phenotypic consequences on McSC quiescence and activation/differentiation and perform mechanistic studies to elucidate the cohorts of genes that they regulate. Understanding how McSCs coordinate these cellular states under homeostasis may shed light onto how these processes are perturbed in disease and help in developing novel therapeutic interventions for conditions such as vitiligo and melanoma.